The objectives of this proposed research are to 1) isolate and characterize arginino-succinate synthetase and argininosuccinate lyase from human liver; 2) compare the properties of the purified hepatic enzymes to those of the enzymes in hon-hepatic tissues and cultured skin fibroblasts; 3) study the residual activity and/or CRM from tissues affected individuals with inherited deficiency of these proteins; 4) perform restriction mapping of synthetase gene in mutant and control cultured skin fibroblasts provided a cloned synthetase gene becomes available. We have isolated the synthetase and lyase from extracts of human liver and have begun characterization of these proteins. We propose to extend these studies to include amino acid analysis, determination of hydrodynamic properties by analytical centrifugation of the native proteins and a more complete kinetic analysis. More specific antibodies have been prepared to each protein. Studies will include comparison of subunit molecular weight in SDS gels, pI determinations, electrophoretic mobility in native gels, and double diffusion analysis. Kinetic properties (Km's) and stability studies will also be conducted. These data will allow for the evaluation of possible tissue specific isoenzymes. Tissues from affected individuals will be studied in an analogous manner after initially determining whether a CRM is present. Cultured skin fibroblasts from normal as well as from patients with inherited deficiency of these proteins will be analyzed for in vitro enzyme activity by sensitive radioisotopic procedures available in our laboratory and also in vivo activity by measuring the ability of cultured cells to incorporate (14C)-citrulline into cellular protein. We will characterize any residual activity in the cell extracts and also analyze CRM in the extracts as described above for tissues. Provided a cloned DNA probe for the argininosuccinate synthetase gene becomes available during this investigation we propose to conduct restriction mapping of the gene in DNA isolated from mutant and normal fibroblasts by Southern blotting experiments.